It is now widely appreciated that bacterial cells have a dramatically complex structural organization, with many individual proteins distributed in the cells in a highly nonuniform pattern that may change rapidly as the cell grows. In the context of bacterial infection and pathogenesis, the dynamic behavior of proteins in the cell envelope is likely to be particularly important, since it is the outside surface of the bacterial cell that directly contacts the host. Technical barriers have made it difficult to directly examine the distribution and mobility of bacterial surface proteins, particularly integral outer membrane proteins in Gram-negative bacterial pathogens. The aim of the project described in this proposal is to exploit several recently developed techniques that allow observation of surface protein dynamics in living bacterial cells to study the role of membrane protein mobility in the persistence and pathogenesis of disease caused by several enteric bacteria, including the category B pathogens Shigella flexneri, Salmonella enterics, Yersinia spp., and enteropathogenic Escherichia coli (EPEC). These new techniques, based on quantitative analysis of videomicroscopy images, are capable of tracing both large-scale protein distributions as they change over time and small-scale movements of individual protein molecules on the bacterial surface. They will be used to answer three specific questions about surface protein mobility in the context of bacterial infection: 1) How does mobility of IcsAA/irG in the outer membrane contribute to protein polarization on the surface of Shigella flexneri? 2) How are the mobility and activity of virulence factors in the outer membrane of Salmonella, Yersinia, and EPEC affected by the lipopolysaccharide remodeling associated with infection? and, 3) What is the organization and dynamic behavior of multidrug resistance (MDR) efflux pumps in Gram-negative bacteria, prior to and during drug exposure? .A fourth goal of this project is to develop a suite of high- throughput, automated computational image/analysis techniques that can facilitate analysis of protein dynamics and cell-to-cell variation in experiments on live bacteria, which we will make freely available to the research community.